Combinatorial Biosynthesis Of Anabaena Phycocyanin β Subunit In Escherichia Coli And Its Activities

Phycocyanin is a kind of natural pigment protein that widely exists in cyanobacteria.It has good fluorescence and biocompatibility.As a natural nutrient,it is a hot topic in experimental research and commercial development.Antioxidant,anti-inflammatory,and anti-cancer biological activities of phycocyanin can be applied in many fields such as functional food,cosmetics,and medical care.The current mainstream method of producing phycocyanin in the industry is to extract and separate the product from artificially cultivated cyanobacteria,but traditional production methods are costly,with high losses and low product purity,resulting in generally high prices of cyanobacterial protein products on the market.With the growing demand,it is extremely important to find ways to efficiently produce phycocyanin.It has been shown that theα-subunit andβ-subunit of natural phycocyanin have excellent antioxidation properties while recombinant phycocyanin has been proven to be structurally identical to natural phycocyanin and has stronger antioxidant capacity.In view of this,this study is based on the synthesis pathway of Anabaena sp.PCC7120 phycocyaninβsubunit,and the key enzyme gene for the synthesis of phycocyaninβsubunit was constructed into E.coli BL21by means of genetic engineering to achieve a heterologous synthesis of phycocyanin.On this basis,the components of the fermentation medium were optimized,and the antioxidant capacity of the recombinant phycocyanin was determined,and its effectiveness as an antioxidant in the application of fruit and vegetable preservation was evaluated.The main research contents were as follows:（1）The recombinant strain E.coli BL21/p ETDuet-1-cpes-cpcb-hox1-pcya strain was successfully constructed and obtained to construct the minimal biosynthetic pathway of Cpc B from heme to phycocyanin.Phycocyaninβsubunit was effectively expressed.The molecular weight of the recombinant protein was about 20 k D after SDS-PAGE electrophoresis analysis.Through spectral detection and analysis,the recombinant protein had a maximum absorption peak at a wavelength of 615 nm and a maximum fluorescence peak at 640 nm,which is basically consistent with theβsubunit of natural Anabaena sp.PCC 7120 phycocyanin.（2）The key components of the fermentation medium of the recombinant strain were successfully optimized by the response surface optimization method.The optimal medium components were obtained by Plackett-Burman tests,steepest climb tests and Box-Behnken tests successively as follows:peptone 12 g/L,yeast extract 9.09 g/L,Na Cl 7.21 g/L,K₂HPO₄10.99 g/L,KH₂PO₄ 4 g/L,Mg SO₄ 1.5 g/L,NH₄Cl 4 g/L,glycerol 6 g/L.The fermentation time of 36 h and final yield of 42.26 mg/L under these conditions were determined by measuring the fermentation curve.（3）The antioxidant activity of recombinant Cpc B was explored,and the antioxidant ability of the purified protein was evaluated by the determination of total reducing power and free radical scavenging rate.The results showed that recombinant Cpc B had strong free radical scavenging ability in a concentration-dependent manner.In the tested experiments,the scavenging effect of recombinant Cpc B was:·OH>ABTS*>DPPH>·O^2-.Further treatment of fresh-cut apples with color difference analysis and browning index showed that recombinant Cpc B could effectively delay the color change of fresh-cut apples for a certain period of time and inhibit the browning of the flesh surface.It shows that recombinant Cpc B has the potential to be developed as a fresh-keeping agent for fruits and vegetables.